Phospholipids and glycosphingolipids are distributed asymmetrically in plasma membrane leaflets, with phosphatidylserine (PS) and phosphatidylethanolamine (PE) in the inner leaflet, and phosphatidylcholine (PC), galactosylceramide (GalCer) and glucosylceramide (GluCer) mainly in the outer leaflet (1,2). The lipid asymmetry is disrupted in various processes, including apoptotic cell death (3), activated platelets (4), red blood cell aging (5), pyrenocyte formation in definitive erythropoiesis (6), fusion of macrophages, myocytes, or cytotrophoblasts (7-9), and sperm capacitation (10).
Distribution of lipids in plasma membranes is regulated by three types of lipid transporters: flippases, floppases and scramblases. Flippases, also called ATP-dependent aminophospholipid translocases, transport aminophospholipids from the extracellular leaflet to the cytoplasmic side (1,11). The type IV-P-type ATPases (P4-ATPase), a subfamily of the P-type ATPase multispan transmembrane proteins, are strong candidates for flippases (12). Floppases are transporters that move a wide range of lipids from the cytosolic to the extracellular leaflet in an ATP-dependent manner. The ATP-binding cassette (ABC) ATPase, particularly ABCA1, has been proposed as a floppase (13), but ABCA1-deficient cells exhibit no defects in transbilayer phospholipid movement (14) arguing against this role.
Once established, the phospholipid distribution between the outer and inner leaflets is not easily disrupted; ATP-dependent translocase inactivation alone does not appear sufficient to cause the rapid PS exposure seen in apoptotic cell death and platelet activation. Thus, a phospholipid scramblase that bi-directionally and non-specifically transports phospholipids in response to Ca2+ has been proposed (15). Using a liposome reconstitution system with synthetic phospholipids, Basse et al. (16) purified a 37-kDa protein from human erythrocytes, and named it phospholipid scramblase (PLSCR). Its cDNA was then isolated (17). However, since the Ca2+-induced PS exposure is normal in PLSCR1−/− cells (18), PLSCR's function as a phospholipid scramblase has been challenged (15,19).
By repeatedly selecting cell populations that efficiently exposed PS in response to Ca2+ ionophore, we recently established a subline of mouse pro B cell line (Ba/F3) that constitutively exposes PS (20). The Ba/F3 subline harbours a mutated form of TMEM16F protein, a protein carrying eight transmembrane regions with cytoplasmic N- and C-termini. Ba/F3 cells carrying the mutated form of TMEM16F constitutively exposed PS and PE, and internalized PC and SM. We thus proposed TMEM16F as a phospholipid scramblase (20). Confirming that TMEM16F is a Ca2+-dependent phospholipid scramblase, recessive TMEM16F mutations were identified in human patients with Scott syndrome (20,21), which is known to result from a phospholipid-scrambling defect; these patients suffer from impaired blood clotting. However, it is not clear if TMEM16F is involved in other processes, such as apoptotic cell death or cell fusion. Two of the TMEM16 family's 10 members, TMEM16A and 16B, are Ca2+-dependent Cl− channels (22-24); this raises a question of whether TMEM16F is likewise a Cl− channel, and whether any other TMEM16 family members are phospholipid scramblases.